Pneumococcal surface protein C (PspC), epitopic regions and strain selection thereof, and uses therefor

ABSTRACT

Disclosed and claimed are: epitopic regions of Pneumococcal Surface Protein C or “PspC”, different clades of PspC, isolated and/or purified nucleic acid molecules such as DNA encoding a fragment or portion of PspC such as an epitopic region of PspC or at least one epitope of PspC, uses for such nucleic acid molecules, e.g., to detect the presence of PspC or of  S. pneumoniae  by detecting a nucleic acid molecule therefor in a sample such as by amplification and/or a polymerase chain reaction, vectors or plasmids which contain and/or express such nucleic acid molecles, e.g., in vitro or in vivo, immunological, immunogenic or vaccine compositions including at least one PspC and/or a portion thereof (such as at least one epitopic region of at least one PspC and/or at least one polypeptide encoding at least one epitope of at least one PspC), either alone or in further combination with at least one second pneumococcal antigen, such as at least one different PspC and/or a fragment thereof and/or at least one PspA and/or at least one epitopic region of at least one PspA and/or at least one polypeptide including at least one epitope of PspA. PspC or a fragment thereof, and thus a composition including PspC or a fragment thereof, can be administered by the same routes, and in approximately the same amounts, as PspA. Thus, the invention further provides methods for administering PspC or a fragment thereof, as well as uses of PspC or a fragment thereof to formulate such compositions.

RELATED APPLICATIONS/PATENTS

This application is a continuation of U.S. patent application Ser. No.09/748,875, filed Dec. 26, 2000, now abandoned, which is a divisionalapplication of Ser. No. 09/298,523, filed on Apr. 23, 1999. Thisapplication is also based upon and claims the benefit of U.S.Provisional Patent Application Ser. No. 60/082,728, filed Apr. 23. 1998.

Reference also is made to: Briles et al., “Strain Selection ofPneumococcal Surface Proteins,” U.S. application Ser. No. 08/710,749,filed Sep. 20, 1996, now U.S. Pat. No. 5,955,089; PCT applicationsPCT/US96/14819, filed Sep. 16, 1996 and WO 97/09994, published Mar. 20,1997; Briles et al. “Oral Administration . . . ,” U.S. application Ser.No. 08/482,981, filed Jun. 7, 1995, now U.S. Pat. No. 6,232,116, U.S.application Ser. No. 08/458,399, filed Jun. 2, 1995, now U.S. Pat. No.6,231,870 and U.S. application Ser. No. 08/657,751, filed May 30, 1996,now U.S. Pat. No. 6,004,802; “Mucosal Administration . . . ,” Briles etal., U.S. application Ser. No. 08/446,201, filed May 19, 1995, now U.S.Pat. No. 6,042,832 (filed as a CIP of U.S. Ser. No. 08/246,636, filedMay 20, 1994, now U.S. Pat. No. 5,965,141) and Briles et al., U.S.application Ser. No. 08/312,949, filed Sep. 30, 1994, now U.S. Pat. No.6,027,734; Briles et al., “Epitopic Regions of Pneumococcal SurfaceProtein A,” U.S. application Ser. No. 08/319,795, filed May 20, 1994 nowU.S. Pat. No. 5,980,909; Briles et al., “Structural Gene of PneumococcalProtein”, U.S. application Ser. No. 08/467,852, filed Jun. 6, 1995, nowU.S. Pat. No. 5,856,170 (filed as a cont. of U.S. application Ser. No.08/247,491, filed May 23, 1994, now U.S. Pat. No. 5,965,400), U.S.application Ser. No. 08/072,070, filed Jun. 3, 1993, now U.S. Pat. No.5,476,929, U.S. application Ser. No. 08/469,434, filed Jun. 6, 1995, nowU.S. Pat. No. 5,753,463 and U.S. application Ser. No. 08/214,164, filedMar. 14, 1994, now U.S. Pat. No. 5,728,387; Briles et al., “TruncatedPspA . . . ,” U.S. application Ser. No. 08/214,222, filed Mar. 17, 1994,now U.S. Pat. No. 5,804,193 and Briles et al., U.S. application Ser. No.08/468,985, now U.S. Pat. No. 5,997,882; Briles et al., “ImmunoassayComprising a Truncated Pneumococcal Surface Protein A (PspA),” U.S.application Ser. No. 08/468,718, filed Jun. 6, 1995, now U.S. Pat. No.5,871,943; U.S. application Ser. No. 08/226,844, filed May 29, 1992;U.S. application Ser. No. 08/093,907, filed Jul. 5, 1994 and 07/889,918,filed Jul. 5, 1994, both abandoned; PCT/US93/05191; and Briles et al.,WO 92/1448.

STATEMENT OF GOVERNMENT SUPPORT

This work was supported in part by National Institute of Health GrantsA121548 and HL58418.

Each of these applications and patents, as well as each document orreference cited in each of these applications and patents (includingduring the prosecution of each issued patent) and PCT and foreignapplications or patents corresponding to and/or claiming priority fromany of the foregoing applications and patents, is hereby expresslyincorporated herein by reference. Documents or references are also citedin the following text, either in a Reference List before the claims, orin the text itself; and, each of these documents or references(“herein-cited documents or references”), as well as each document orreference cited in each of the herein-cited documents or references, ishereby expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to epitopic regions of PneumococcalSurface Protein C or “PspC”, different clades of PspC, isolated and/orpurified nucleic acid molecules, such as DNA encoding a fragment orportion of PspC such as an epitopic region of PspC or at least oneepitope of PspC, uses for such nucleic acid molecules, e.g., to detectthe presence of PspC or of Streptococcus pneumoniae by detecting anucleic acid molecule therefor in a sample such as by amplificationand/or a polymerase chain reaction, vectors or plasmids which containand/or express such nucleic acid molecules, e.g, in vitro or in vivo,immunological, immunogenic or vaccine compositions comprising at leastone PspC and/or a portion thereof (such as at least one epitopic regionof at least one PspC and/or at least one polypeptide encoding at leastone epitope of at least one PspC), either alone or in furthercombination with at least one second pneumococcal antigen, such as atleast one different PspC and/or a fragment thereof and/or at least onePspA and/or at least one epitopic region of at least one PspA and/or atleast polypeptide comprising at least one epitope of PspA.

PspC or a fragment thereof, and thus a composition comprising PspC or afragment thereof, can be administered by the same routes, and inapproximately the same amounts, as PspA. Thus, the invention furtherprovides methods for administering PspC or a fragment thereof, as wellas uses of PspC or a fragment thereof to formulate such compositions.

Other aspects of the invention are described in or are obvious from (andwithin the ambit of the invention) the following disclosure.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is an important cause of otitis media,meningitis, bacteremia and pneumonia, and a leading cause of fatalinfections in the elderly and persons with underlying medicalconditions, such as pulmonary disease, liver disease, alcoholism, sicklecell anemia, cerebrospinal fluid leaks, acquired immune deficiencysyndrome (AIDS), and in patients undergoing immunosuppressive therapy.It is also a leading cause of morbidity in young children. Pneumococcalinfections cause approximately 40,000 deaths in the U.S. yearly. Themost severe pneumococcal infections involve invasive meningitis andbacteremia infections, of which there are 3,000 and 50,000 casesannually, respectively.

Despite the use of antibiotics and vaccines, the prevalence ofpneumococcal infections has declined little over the last twenty-fiveyears; the case-fatality rate for bacteremia is reported to be 15–20% inthe general population, 30–40% in the elderly, and 36% in inner-cityAfrican Americans. Less severe forms of pneumococcal disease arepneumonia, of which there are 500,000 cases annually in the U.S., andotitis media in children, of which there are an estimated 7,000,000cases annually in the U.S. caused by pneumococcus. Strains ofdrug-resistant S. pneumoniae are becoming ever more common in the U.S.and worldwide. In some areas, as many as 30% of pneumococcal isolatesare resistant to penicillin. The increase in antimicrobial resistantpneumococcus further emphasizes the need for preventing pneumococcalinfections.

Pneumococcus asymptomatically colonizes the upper respiratory tract ofnormal individuals; disease often results from the spread of organismsfrom the nasopharynx to other tissues during opportunistic events. Theincidence of carriage in humans varies with age and circumstances.Carrier rates in children are typically higher than those of adults.Studies have demonstrated that 38 to 60% of preschool children, 29 to35% of grammar school children and 9 to 25% of junior high schoolchildren are carriers of pneumococcus. Among adults, the rate ofcarriage drops to 6% for those without children at home, and to 18 to29% for those with children at home. It is not surprising that thehigher rate of carriage in children than in adults parallels theincidence of pneumococcal disease in these populations.

An attractive goal for streptococcal vaccination is to reduce carriagein the vaccinated populations and subsequently reduce the incidence ofpneumococcal disease. There is speculation that a reduction inpneumococcal carriage rates by vaccination could reduce the incidence ofthe disease in non-vaccinated individuals as well as in vaccinatedindividuals. This “herd immunity” induced by vaccination against upperrespiratory bacterial pathogens has been observed using the Haemophilusinfluenzae type b conjugate vaccines (Takala, A. K., et al., J. Infect.Dis. 1991; 164: 982–986; Takala, A. K., et al., Pediatr. Infect. Dis.J., 1993; 12: 593–599; Ward, J., et al., Vaccines, S. A. Plotkin and E.A. Mortimer, eds., 1994, pp. 337–386; Murphy, T. V., et al., J.Pediatr., 1993; 122; 517–523; and Mohle-Boetani, J. C., et al., Pediatr.Infect. Dis. J., 1993; 12: 589–593).

It is generally accepted that immunity to S. pneumoniae can be mediatedby specific antibodies against the polysaccharide capsule of thepneumococcus. However, neonates and young children fail to make anadequate immune response against most capsular polysaccharide antigensand can have repeated infections involving the same capsular serotype.One approach to immunizing infants against a number of encapsulatedbacteria is to conjugate the capsular polysaccharide antigens to proteinto make them immunogenic. This approach has been successful, forexample, with H. influenzae b (see U.S. Pat. No. 4,496,538 to Gordon andU.S. Pat. No. 4,673,574 to Anderson).

However, there are over ninety known capsular serotypes of S.pneumoniae, of which twenty-three account for about 95% of the disease.For a pneumococcal polysaccharide-protein conjugate to be successful,the capsular types responsible for most pneumococcal infections wouldhave to be made adequately immunogenic. This approach may be difficult,because the twenty-three polysaccharides included in thepresently-available vaccine are not all adequately immunogenic, even inadults.

Protection mediated by anti-capsular polysaccharide antibody responsesare restricted to the polysaccharide type. Different polysaccharidetypes differentially facilitate virulence in humans and other species.Pneumococcal vaccines have been developed by combining 23 differentcapsular polysaccharides that are the prevalent types of humanpneumococcal disease. These 23 polysaccharide types have been used in alicensed pneumococcal vaccine since 1983 (D. S. Fedson and D. M. Musher,Vaccines, S. A. Plotkin and J. E. A. Montimer, eds., 1994, pp. 517–564).The licensed 23-valent polysaccharide vaccine has a reported efficacy ofapproximately 60% in preventing bacteremia caused pneumococci in healthyadults.

However, the efficacy of the vaccine has been controversial, and attimes, the justification for the recommended use of the vaccinequestioned. It has been speculated that the efficacy of this vaccine isnegatively affected by having to combine 23 different antigens. Having alarge number of antigens combined in a single formulation may negativelyaffect the antibody responses to individual types within this mixturebecause of antigenic competition. The efficacy is also affected by thefact that the 23 serotypes encompass all serological types associatedwith human infections and carriage.

An alternative approach to protecting against pneumococcal infection,especially for protecting children, and also the elderly, would be toidentify protein antigens that could elicit protective immune responses.Such proteins may serve as a vaccine by themselves, may be used inconjunction with successful polysaccharide-protein conjugates, or ascarriers for polysaccharides.

Pneumococcal Surface Protein A or PspA has been identified as anantigen; and, its DNA and amino acid sequences have been investigated.PspA is useful in eliciting protective immune responses. PspA orfragments thereof can be used in immunological, immunogenic or vaccinecompositions; and, such compositions can contain different types ofPspAs or fragments from different types of PspAs. Further, suchcompositions can be administered by injection, or mucosally or orally,or by means of a vector expressing the PspA or fragment thereof.

Studies on PspA led to the discovery of a PspA-like protein and apspA-like gene, now termed PspC and pspC. Indeed, early patentliterature termed PspC as “PspA-like”.

It is believed that heretofore that epitopic regions of PspC have notbeen disclosed or suggested. It is likewise believed that heretoforedifferent clades of PspC have not been taught or suggested. Further, itis believed that heretofore DNA encoding epitopic regions of PspC havenot been disclosed or suggested. Further still, it is believed thatheretofore immunological, immunogenic or vaccine compositions comprisingat least one PspC and/or portions thereof (such as at least one epitopicregion of at least one PspC and/or at least one polypeptide encoding atleast one epitope of at least one PspC), either alone or in furthercombination with at least one second pneumococcal antigen, such as atleast one different PspC and/or a fragment thereof and/or at least onePspA and/or at least one epitopic region of at least one PspA and/or atleast one polypeptide comprising at least one epitope of PspA, have notbeen taught or suggested.

Alternative vaccination strategies are desirable as such providealternative immunological, immunogenic or vaccine compositions, as wellas alternative routes to administration or alternative routes toresponses. It would be advantageous to provide an immunologicalcomposition or vaccination regimen which elicits protection againstvarious diversified pneumococcal strains, without having to combine alarge number of possibly competitive antigens within the sameformulation. And, it is advantageous to provide additional antigens andepitopes for use in immunological, immunogenic and/or vaccinecompositions, e.g., to provide alternative compositions containing orcomprising such antigens or epitopes either alone or in combination withdifferent antigens.

Furthermore it is advantageous to provide a better understanding of thepathogenic mechanisms of pneumococci, as this can lead to thedevelopment of improved vaccines, diagnoses and treatments.

OBJECTS AND SUMMARY OF THE INVENTION

An object of the invention can include providing one or more of:epitopic regions of PspC, different clades of PspC, isolated and/orpurified nucleic acid molecules such as DNA encoding a fragment orportion of PspC such as an epitopic region of PspC or at least oneepitope of PspC, uses for such nucleic acid molecules, vectors orplasmids which contain and/or express such nucleic acid molecules, e.g.,in vitro or in vivo, immunological, immunogenic or vaccine compositionscomprising such a vector or plasmid and/or at least one PspC and/or aportion thereof (such as at least one epitopic region of at least onePspC and/or at least one polypeptide encoding at least one epitope of atleast one PspC), either alone or in further combination with at leastone second pneumococcal antigen, such as at least one different PspCand/or a fragment thereof and/or at least one PspA and/or at least oneepitopic region of at least one PspA and/or at least one polypeptidecomprising at least one epitope of PspA and/or at least one vector orplasmid expressing said second pneumococcal antigen (which vector orplasmid could be the same as the aforementioned vector or plasmidcomprising a nucleic acid molecule encoding PspC or a portion orfragment thereof); and, methods for administering PspC or a fragmentthereof, as well as uses of PspC or a fragment thereof to formulate suchcompositions, inter alia.

Accordingly, the invention can provide one or more of: epitopic regionsof PspC, different clades of PspC, isolated and/or purified nucleic acidmolecules such as DNA encoding a fragment or portion of PspC such as anepitopic region of PspC or at least one epitope of PspC, uses for suchnucleic acid molecules, vectors or plasmids which contain and/or expresssuch nucleic acid molecules, e.g., in vitro or in vivo, immunological,immunogenic or vaccine compositions comprising such a vector or plasmidand/or at least one PspC and/or a portion thereof (such as at least oneepitopic region of at least one PspC and/or at least one polypeptideencoding at least one epitope of at least one PspC), either alone or infurther combination with at least one second pneumococcal antigen, suchas at least one different PspC and/or a fragment thereof and/or at leastone PspA and/or at least one epitopic region of at least one PspA and/orat least one polypeptide comprising at least one epitope of PspA and/orat least one vector or plasmid expressing said second pneumococcalantigen (which vector or plasmid could be the same as the aforementionedvector or plasmid comprising a nucleic acid molecule encoding PspC or aportion or fragment thereof); and, methods for administering PspC or afragment thereof, as well as uses of PspC or a fragment thereof toformulate such compositions, inter alia.

PspC or a fragment thereof, and thus a composition comprising PspC or afragment thereof, can be administered by the same routes, and inapproximately the same amounts, as PspA. Thus, the invention furtherprovides methods for administering PspC or a fragment thereof, as wellas uses of PspC or a fragment thereof to formulate such compositions.

Still further, the invention provides PspC epitopic regions, e.g., thealpha helical region, or the proline region or the combination of thealpha helical and proline regions, or the entire PspC molecule, or aa1–590 of PspC clade A, or amino acid(s) (“aa”) 1–204 or aa 46–204 or aa1–295 or aa 46–295 or aa 1–454 or aa 46–454 or aa 204–454 or aa 295–454or aa 1–590 or aa 46–590 or aa 204–590 or aa 295–590 or aa 454–590 or aa1–652 or aa 46–652 or aa 204–652 or aa 295–652 or aa 454–652 or aa590–652 or aa 1–892 or aa 46–892 or aa 204–892 or aa 295–892 or aa454–892 or 590–892 of PspC clade A. A prototypic clade A PspC isPspC.EF6796. In other clade A PspCs, the epitopic regions may haveslightly different amino acid numbers. Thus, the invention comprehendsregions of other clade A PspCs which are substantially homologous, orsignificantly homologous, or highly homologous, or very highlyhomologous, or identical, or highly conserved, with respect to theforegoing particularly recited epitopic regions. Also, where possible,these regions can extend in either the N-terminal or COOH-terminaldirection; e.g., by about another 1–25 or 1–50 amino acids in either orboth directions. The invention further provides a polypeptide comprisingat least one epitopic region or at least one epitope in any one of thesevarious regions.

Similarly, the invention provides clade b epitopic regions, e.g., thealpha helical region, the proline region, the combination of the alphahelical and proline regions, and the entire molecule, as well as by aasuch as aa 1–664, or aa 1–375, or aa 1–445 or aa 1–101, or aa 1–193, oraa 1–262, or aa 1–355, or aa 101–193, or aa 101–262, or aa 101–355, oraa 101–375, or aa 101–455 or aa 193–262, or aa 193–355, or aa 193–375,or aa 193–445 or aa 262–355, or aa 262–375, or aa 262–445 or aa 355–375,or aa 355–445 or aa 375–445 or aa 101–664, or aa 193–664, or aa 262–664,or aa 355–664 or aa 375–664 or aa 1-end of proline subregion A, or aa1-beginning of proline subregion B, or aa 101-end of proline subregionA, or aa 101-beginning of proline subregion B, or aa 193-end of prolinesubregion A, or aa 193-beginning of proline subregion B, or aa 262-endof proline subregion A, or aa 262-beginning of proline subregion B, oraa 355-end of proline subregion A, or aa 355-beginning of prolinesubregion B, or aa 375-end of proline subregion A, or proline subregionA, or aa 375-beginning of proline subregion B, or proline subregion B,or beginning of proline subregion B-aa 664. A prototypic clade b PspC isPspC.D39. In other clade B PspCs, the epitopic regions may have slightlydifferent amino acid numbers. Thus, the invention comprehends regions ofother clade b PspCs which are substantially homologous, or significantlyhomologous, or highly homologous, or very highly homologous, oridentical, or highly conserved, with respect to the foregoingparticularly recited epitopic regions. Also, where possible, theseregions can extend in either the N-terminal or COOH-terminal direction;e.g., by about another 1–25 or 1–50 amino acids in either or bothdirections. For instance, interesting epitopic regions include: aa263–482, 1–445 and 255–445. And, the invention further provides apolypeptide comprising at least one epitopic region or at least oneepitope in any one of these regions.

A polypeptide comprising at least one epitope of PspC or PspA can beshorter than natural or full length PspC or PspA, e.g., a truncated PspCor PspA, such as comprising up to about 90% of natural or full lengthPspC or PspA.

The invention further provides an isolated nucleic acid molecule, e.g.,DNA comprising a sequence encoding any one of these epitopic regions ora polypeptide comprising at least one of these epitopic regions, or anepitope of PspC; such a nucleic acid molecule is advantageously at leastabout 12 nucleotides in length, for instance, at least about 15, about18, about 21, about 24 or about 27 nucleotides in length, such as atleast about 30, about 33, about 36, about 39 or about 42 nucleotides inlength, for example, a nucleic acid molecule of at least about 12nucleotides in length such as about 12 to about 30, about 12 to about 50or about 12 to about 60, or about 12 to about 75 or about 12 to about100 or more nucleotides in length. A nucleic acid molecule comprising asequence encoding at least one epitope of PspC or PspA can be shorterthan natural or full length pspC or pspA, e.g., a truncated pspC orpspA, such as comprising up to about 90% of natural or full length pspCor pspA or encoding up to about 90% of natural or full length PspA orPspC.

Moreover, in this disclosure, Applicants demonstrate cross-reactivitybetween PspC and PspA, as well as regions of PspC and PspA and/or ofpspC and pspA which are highly conserved, substantially homologous,highly homologous, and identical. This information allows the skilledartisan to identify nucleic acid molecules which can hybridize, e.g.,specifically (“specific hybridization”), to pspC or pspA or both pspCand pspA, e.g., under stringent conditions. The term “specifichybridization” will be understood to mean that the nucleic acid probesof the invention are capable of stable, double-stranded hybridization tobacterially-derived DNA or RNA under conditions of high stringency, asthe term “high stringency” would be understood by those with skill inthe art (see, for example, Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. and Hames and Higgins, eds., 1985, Nucleic AcidHybridization, IRL Press, Oxford, U.K.). Hybridization will beunderstood to be accomplished using well-established techniques,including but not limited to Southern blot hybridization, Northern blothybridization, in situ hybridization and, most preferably, Southernhybridization to PCR-amplified DNA fragments. In a preferredalternative, the nucleic acid hybridization probe of the invention maybe obtained by use of the polymerase chain reaction (PCR) procedure,using appropriate pairs of PCR oligonucleotide primers as providedherein or from the teachings herein. See U.S. Pat. No. 4,683,195 toMullis et al. and U.S. Pat. No. 4,683,202 to Mullis. A probe or primercan be any stretch of at least 8, preferably at least 10, morepreferably at least 12, 13, 14, or 15, such as at least 20, e.g., atleast 23 or 25, for instance at least 27 or 30 nucleotides in pspC whichare unique to pspC, e.g., not also in pspA (when amplification of justpspC is desired) or unique to both pspC and pspA or in both pspC andpspA (when amplification of both is acceptable or desired) or which arein pspC and are least conserved among the pspC/pspA genes. As to PCR orhybridization primers or probes and optimal lengths therefor, referenceis also made to Kajimura et al., GATA 7(4):71–79 (1990). The inventionwill thus be understood to provide oligonucleotides, such as, pairs ofoligonucleotides, for use as primers for the in vitro amplification ofbacterial DNA samples and fragments thereof, or for use in expressing aportion of bacterial DNA, either in vitro or in vivo. Theoligonucleotides preferably specifically hybridize to sequences flankinga nucleic acid to be amplified, wherein the oligonucleotides hybridizeto different and opposite strands of the double-stranded DNA target. Theoligonucleotides of the invention are preferably derived from thenucleic acid molecules and teachings herein. As used in the practice ofthis invention, the term “derived from” is intended to encompass thedevelopment of such oligonucleotides from the nucleic acid molecules andteachings disclosed herein, from which a multiplicity of alternative andvariant oligonucleotides can be prepared.

And, the invention further comprehends vectors or plasmids containingand/or expressing such a nucleic acid molecule, as well as uses of suchnucleic acid molecules, e.g., for expression of PspC or an epitopicregion thereof or at least an epitope thereof or a polypeptidecomprising at least one epitope thereof either in vitro or in vivo, orfor amplifying or detecting PspC or S. pneumoniae in a sample, forinstance by a PCR.

With respect to the herein mentioned nucleic acid molecules andpolypeptides, e.g., the aforementioned nucleic acid molecules andpolypeptides, the invention further comprehends isolated and/or purifiednucleic acid molecules and isolated and/or purified polypeptides havingat least about 70%, preferably at least about 75% or about 77% identityor homology (“substantially homologous or identical”), advantageously atleast about 80% or about 83%, such as at least about 85% or about 87%homology or identity (“significantly homologous or identical”), forinstance at least about 90% or about 93% identity or homology (“highlyhomologous or identical”), more advantageously at least about 95%, e.g.,at least about 97%, about 98%, about 99% or even about 100% identity orhomology (“very highly homologous or identical” to “identical”; or fromabout 84–100% identity considered “highly conserved”). The inventionalso comprehends that these nucleic acid molecules and polypeptides canbe used in the same fashion as the herein or aforementioned nucleic acidmolecules and polypeptides.

Nucleotide sequence homology can be determined using the “Align” programof Myers and Miller, (“Optimal Alignments in Linear Space”, CABIOS,4:11–17, 1988, incorporated herein by reference) and available at NCBI.Alternatively or additionally, the terms “homology” or “identity”, forinstance, with respect to a nucleotide or amino acid sequence, canindicate a quantitative measure of homology between two sequences. Thepercent sequence homology can be calculated as(N_(ref)−N_(dif))*100/N_(ref), wherein N_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinN_(ref) is the number of residues in one of the sequences. Hence, theDNA sequence AGTCAGTC will have a sequence similarity of 75% with thesequence AATCAATC (N_(ref)=8; N_(dif)=2).

Alternatively or additionally, “homology” or “identity” with respect tosequences can refer to the number of positions with identicalnucleotides or amino acids divided by the number of nucleotides or aminoacids in the shorter of the two sequences wherein alignment of the twosequences can be determined in accordance with the Wilbur and Lipmanalgorithm (Wilbur and Lipman, 1983 PNAS USA 80:726, incorporated hereinby reference), for instance, using a window size of 20 nucleotides, aword length of 4 nucleotides, and a gap penalty of 4, andcomputer-assisted analysis and interpretation of the sequence dataincluding alignment can be conveniently performed using commerciallyavailable programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc.CA). When RNA sequences are said to be similar, or have a degree ofsequence identity or homology with DNA sequences, thymidine (T) in theDNA sequence is considered equal to uracil (U) in the RNA sequence.

RNA sequences within the scope of the invention can be derived from DNAsequences, by thymidine (T) in the DNA sequence being considered equalto uracil (U) in RNA sequences.

Additionally or alternatively, amino acid sequence similarity oridentity or homology can be determined using the BlastP program(Altschul et al., Nucl. Acids Res. 25: 3389–3402, incorporated herein byreference) and available at NCBI. The following references (eachincorporated herein by reference) provide algorithms for comparing therelative identity or homology of amino acid residues of two proteins,and additionally or alternatively with respect to the foregoing, theteachings in these references can be used for determining percenthomology or identity: Needleman S b and Wunsch C D, “A general methodapplicable to the search for similarities in the amino acid sequences oftwo proteins,” J. Mol. Biol. 48:444–453 (1970); Smith T F and Waterman MS, “Comparison of Bio-sequences,” Advances in Applied Mathematics2:482–489 (1981); Smith T F, Waterman M S and Sadler J R, “Statisticalcharacterization of nucleic acid sequence functional domains,” Nucl.Acids Res., 11:2205–2220 (1983); Feng D F and Dolittle R F, “Progressivesequence alignment as a prerequisite to correct phylogenetic trees,” J.Molec. Evol., 25:351–360 (1987); Higgins D G and Sharp P M, “Fast andsensitive multiple sequence alignment on a microcomputer,” CABIOS, 5:151–153 (1989); Thompson J D, Higgins D G and Gibson T J, “Cluster W:improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighing, positions specific gap penalities and weightmatrix choice, Nucl. Acids Res., 22:4673–480 (1994); and, Devereux J,Haeberlie P and Smithies O, “A comprehensive set of sequence analysisprogram for the VAX, Nucl. Acids Res., 12: 387–395 (1984).

A polypeptide comprising at least a fragment or epitope of PspC, e.g.,an epitopic region of PspC or PspC, can be a fusion protein; forinstance, fused to a protein which enhances immunogenicity, such as aCholera Toxin, e.g., Cholera Toxin b (CTB).

Similarly, a polypeptide comprising at least a fragment or epitope ofPspC, e.g., an epitopic region of PspC or PspC, can be administered withan adjuvant or a vehicle which enhances immunogenicity, such as CTB.

Thus, the invention provides an immunological, immunogenic or vaccinecomposition comprising at least one PspC and/or a portion thereof (suchas at least one epitopic region of at least one PspC and/or at least onepolypeptide encoding at least one epitope of at least one PspC), eitheralone or in further combination with at least one second pneumococcalantigen, such as at least one different PspC and/or a fragment thereofand/or at least one PspA and/or at least one epitopic region of at leastone PspA and/or at least one polypeptide comprising at least one epitopeof PspA. The epitopic region of PspA can be as in applications citedunder “Related Applications”, supra, e.g., aa 1 to 115, 1 to 314, 1 to260, 192 to 260, 192 to 588, 192 to 299, 1–301, 1–314 or 1–370 of PspA.From the teachings herein and in the applications cited under “RelatedApplications”, the skilled artisan can select an epitope of interest,e.g, of PspC and/or PspA.

This invention also provides strain selection of PspCs from strains forvaccine compositions, based upon sequence homology and cross-reactivity,akin to that which Applicants have done with PspA. PspC strains can beclassified according to sequence homology in the alpha helical and/orproline rich regions, and assigned to a clade, and subsequently, eachclade is assigned to a family. Applicants have thus determined that sofar there is at least one PspC family with at least two major clades.

Inventive compositions, such as immunogenic, immunological or vaccinecompositions, can comprise at least one PspC (or immunogenic fragmentthereof or polypeptide comprising at least one PspC epitope or epitopicregion or at least one vector or plasmid expressing such PspC orfragment thereof, or at least one PspC epitope or epitopic region),preferably at least two (2), for instance up to ten (10), from strainsfrom each clade (and/or family), alone, or in further combination withat least one PspA (or immunogenic fragment thereof or polypeptidecomprising at least one PspA or at least one epitope or epitopic regionof PspA or at least one vector or plasmid expressing such PspA orfragment thereof, or at least one PspA epitope or epitopic region, whichvector or plasmid can be the same as the aforementioned vector orplasmid) or preferably at least two (2), for instance up to ten (10),from strains from each PspA clade (and/or family), for a broadlyefficacious pneumococcal vaccine with a limited number of strains.

Immunogenic, immunological or vaccine compositions of the invention canbe administered in the same ways as PspA immunogenic, immunological orvaccine compositions, e.g., by injection, mucosally, orally, nasally,and the like, and/or by way of in vivo expression thereof by a plasmidor vector, as well as in same or similar regimens (e.g., such as byprime boost) (see applications cited under Related Applications, as wellas documents cited herein). (Thus, there can be PspA, an epitopic regionof PspA, a polypeptide comprising an epitope within an epitopic regionof PspA, an immunogenic, immunological or vaccine composition comprisingat least one PspA and/or at least one fragment or portion thereof, e.g.,an epitopic region thereof or a polypeptide comprising at least oneepitope from PspA and/or a vector or plasmid expressing a nucleic acidmolecule encoding PspA or a fragment or portion thereof, administrationof PspA or such a polypeptide or such a composition by injection,mucosally, nasally, orally, and the like and/or as part of a prime-boostregimen with another antigen which can also be PspA.) The amount of PspCin such compositions can be analogous to the amount of PspA in PspAimmunogenic, immunological or vaccine compositions (see applicationscited under Related Applications). (Accordingly, there can be PspC, anepitopic region of PspC, a polypeptide comprising an epitope within anepitopic region of PspC, an immunogenic, immunological or vaccinecomposition comprising at least one PspC and/or at least one fragment orportion thereof, e.g., an epitopic region thereof or a polypeptidecomprising at least one epitope from PspC and/or a vector or plasmidexpressing a nucleic acid molecule encoding PspC or a fragment orportion thereof, administration of PspC or such a polypeptide or such acomposition by injection, mucosally, nasally, orally, and the likeand/or as part of a prime-boost regimen with another antigen which canalso be PspC.)

Such compositions are useful in eliciting an immune response in ananimal or a host, such as a protective immune response; or, forgenerating antibodies, which can be subsequently used in kits, tests orassays for detecting the presence of PspC and/or PspA and PspC and/or S.pneumoniae.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF FIGURES

The following Detailed Description, given by way of example, and notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying Figures, incorporatedherein by reference, in which:

FIG. 1 shows a schematic representation of the PspC clade A and clade Band PspA polypeptides in comparison with each other (long arrowsrepresent direct repeats found within the alpha helix; the hypervariableregion is indicated by zig-zag lines; and the region of homology of pspCwith pspA found within the alpha helix is indicated by horizontallines);

FIGS. 2A to 2D show the alignment of PspCs (SEQ ID NOs: 1 to 13) (theamino acid sequences which include the α helical region and theproline-rich region of PspC were aligned using MacVector 6.0; the directrepeats within the α helix, the non-coiled-coil block, and theproline-rich region are indicated with arrows; conserved regions areshaded, and gaps are shown with a dash (-); taxons are named for thestrain from which the gene was cloned with the exception of Genbankentrees: SpsA1 (Y10818) from strain ATCC33400 (serotype 1), SpsA2(AJ002054) from strain ATCC11733 (serotype 2), SpsA47 (AJ002055) fromstrain NCTC10319 (serotype 47), CbpA (AF019904) from strain LM91(serotype 2), C3 bp (AF067128), and tigr from a serotype 4 clinicalisolate; the capsular serotypes of the other strains are as follows:EF6796 (6A), BG8090 (19), L81905 (4), DBL6A (6A), BG9163 (6B), D39 (2)and E134 (23));

FIGS. 3A to 3B show the coiled-coil motif of the alpha-helix of PspC(amino acids that are not in the coiled-coil motif are in the rightcolumn; this is the output from the Matcher program) (SEQ ID NO: 14);

FIG. 4 shows a tree of the PspC proteins from this disclosure andrelated proteins SpsA and CbpA from Genbank (PspC proteins weretruncated after the proline-rich region (FIG. 1) before being alignedusing the ClustalW algorithm and the Blosum30 amino acid scoring matrixin MacVector; the tree is an unrooted phylogram generated by theneighbor-joining method using mean character distances in the programPAUP4.0b (Swofford); non-italic numbers on the tree indicate distancesalong the branch lengths as calculated by PAUP; italic bolded numbersindicate the percentage of time each branch was joined together underbootstrap analysis (1000 replicates performed); Clade A and Clade b areeach monophyletic groups separated by greater than 0.1 distance whichclustered together 100% of the time; Clade A PspC proteins share a 120amino acid domain with many PspA proteins (FIG. 2); Clade b proteinslack the 120 AA domain, but all PspC/SpsA/CbpA proteins share theproline-rich domain with PspA proteins; the boxed D39-lineage indicatesdifferent sequences for this locus originating from strains that arelaboratory descendents of the strain D39; the taxons used were the sameas those described for FIG. 2);

FIGS. 5A to 5B show PspC and PspA consensus of the choline bindingregion (SEQ ID NOs: 15–52);

FIG. 6 shows the reactivity of the PspC antiserum with selectedpneumococcal lysates run in a Western immunoblot;

FIG. 7 shows the level of antibody reactivity to PspC and PspA fragmentspresent in the sera of mice immunized with PspC (each bar represents themean of the log reciprocal titer and upperbound of the standard error ofsera from five mice; the limit of detection of the log reciprocalantibody titer is 1.8);

FIGS. 8A to 8E show amino acid and DNA sequences for SpsA and spsA fromGenbank (SEQ ID NOs: 53 and 54) (accession CAA05158; AJ002054.1;AJ002054; Hammerschmidt et al. 1997);

FIGS. 9A to 9E show an additional amino acid and DNA sequences for SpsAand spsA from Genbank (SEQ ID NOs: 55 and 56) (accession CAA05159;AJ002055; AJ002055.1; Hammerschmidt et al. 1997);

FIGS. 10A to 10E show amino acid and DNA sequences for CbpA and cbpAfrom Genbank (SEQ ID NOS: 57 and 58)(accession AAB70838; AF019904;AF019904.1; Rosenow et al. 1997);

FIGS. 11A to 11F show amino acid and DNA sequences for PspC and pspCfrom Genbank (SEQ ID NOs: 59 and 60)(from EF6796; accession AAD00184;U72655.1; U72655; Brooks-Walter et al.);

FIG. 12 shows a tree of PspC proteins from this disclosure from theUniversity of Alabama, analogous to the tree shown in FIG. 4 (PspCproteins sequenced at the University of Alabama; PspC proteins weretruncated after the proline-rich region—see FIG. 1—before aligned usingthe ClustalW algorithm and the Blosum30 amino acid scoring matrix inMacVector; the tree is an unrooted phylogram generated by theneighbor-joining method using mean character distances in the programPAUP4.0b (Swofford); non-italic numbers on the tree indicate distancesalong the branch lengths as calculated by PAUP; italic bolded numbersindicate the percentage of time each branch was joined together underbootstrap analysis (1000 replicates performed); Clade A and Clade b aremonophyletic groups separated by greater than 0.1 distance whichclustered together 100% of the time; Clade A PspC proteins share a 120amino acid domain with many PspA proteins—see FIG. 2; taxons are namedfor the strain from which the gene was cloned, with the capsularserotypes as follows—EF6796 (6A), BG8090 (19), L81905 (4), DBL6A (6a),BG9163 (6B), D39 (2) and E134 (23));

FIGS. 13A to 13C show the alignment of PspCs (SEQ ID NOs: 61–67) fromthis disclosure from the University of Alabama, analogous to thealignment shown in FIG. 2;

FIG. 14 shows a dendrogram showing the distance of a divergent PspC(from other PspCs), indicating that it likely belongs to a second family(Dendrogram of the PspC/SpsA/Cbpa from Genbank and nearest relativegenes from other species; PspC proteins were truncated after theproline-rich region—see FIG. 1—before being aligned using the ClustalWalgorithm and the Blosum30 amino acid scoring matrix in MacVector; thedendrogram is the guide tree used in alignment by MacVector; smallnumbers on the tree indicate distances along the branch lengths ascalculated during the ClustalW alignment; sequences of two proteins fromStreptococcus agalactiae bac and rib, and one from Enterococcus facaelisare included for comparison; the PspC.V26 is a highly divergent PspCprotein from S. pneumoniae strain V26);

FIGS. 15A to 15C show the amino acid and DNA sequences (SEQ ID NOs: 68to 69) of the divergent PspC (PspC from S. pneumoniae strain V26);

FIGS. 16A–b show the DNA sequence of PspC from strain E134 (SEQ ID NO:70).

FIGS. 17A–b show the DNA sequence of PspC from strain D39 (SEQ ID NO:71).

FIGS. 18A–b show the DNA sequence of PspC from strain BG9163 (SEQ ID NO:72).

FIG. 19 shows the DNA sequence of PspC from strain BG8090 (SEQ ID NO:73).

FIG. 20 shows the DNA sequence of PspC from strain L81905 (SEQ ID NO:74).

FIG. 21 shows the DNA sequence of PspC from strain DBL6a (SEQ ID NO:75).

DETAILED DESCRIPTION

PspC (see FIGS. 1, 2, 3, 4, 5, 11, 12, 13, 14, 15) is one of threedesignations for a pneumococcal surface protein which is PspA-like, andwhose gene is present in approximately 75% of all S. pneumoniae.Applicants have cloned and sequenced the pspC gene and have expressedthe PspC protein (See, e.g., FIGS. 1, 2, 4, 5, 11, 12, 13, and patentapplications cited under the heading Related Applications, supra, aswell as to articles or literature cited herein; see also FIGS. 14, 15).Under the designation SpsA (see FIGS. 8, 9), PspC has been shown to bindsecretory IgA (Hammerschmidt et al. 1997). Under the designation CbpA(see FIG. 10), PspC has been shown to interact with human epithelial andendothelial cells (Rosenow et al. 1997).

The pspC gene is paralogous to the pspA gene in S. pneumoniae and wasthus called pspC (Brooks-Walter et al. 1997; see also applications citedin Related Applications, supra).

The present invention provides epitopic regions of PspC, differentclades of PspC, DNA encoding epitopic regions of PspC, vectors whichexpress such epitopic regions, immunological, immunogenic or vaccinecompositions comprising at least one PspC and/or a portion thereof (suchas at least one epitopic region of at least one PspC and/or at least onepolypeptide encoding at least one epitope of at least one PspC), eitheralone or in further combination with at least one second pneumococcalantigen, such as at least one different PspC and/or a fragment thereofand/or at least one PspA and/or at least one epitopic region of at leastone PspA and/or at least one polypeptide comprising at least one epitopeof PspA.

PspC or a fragment thereof, and thus a composition comprising PspC or afragment thereof, can be administered by the same routes, and inapproximately the same amounts, as PspA. Thus, the invention furtherprovides methods for administering PspC or a fragment thereof or apolypeptide comprising at least one epitope of PspC, as well as uses ofPspC or a fragment thereof to formulate such compositions.

Furthermore, in this disclosure, pspC genes from seven differentclinical S. pneumoniae strains were cloned and sequenced. Examination ofthe sequences of twelve alleles reveals that this gene exists in diverseforms among pneumococci and has a mosaic structure in which sequencemodules encoding protein domains have contributed to the pattern ofvariation during gene evolution.

Two major clades exist: clade A alleles are larger and contain an extramodule that is shared by many pspA genes; clade b alleles are smallerand lack this pspA-like domain. All genes in both clade A and clade bmaintain a proline-rich domain and a choline-binding repeat domain thatare indistinguishable from similar domains in the pspA gene at thenucleotide and protein level.

Thus, this invention also relates to strain selection of PspCs fromstrains for vaccine compositions, based upon sequence homology andcross-reactivity, akin to that which Applicants have done with PspA.PspC strains can be classified according to sequence homology in thealpha helical and/or proline rich regions, and assigned to a clade, andsubsequently, each clade is assigned to a family. Applicants have thusdetermined that so far there is one PspC family with at least two majorclades.

There is, however, a single PspC (PspC.V26, from S. pneumoniae strainV26, a capsular-type 14 S. pneumoniae strain) that appears to be amember of a second family because it seems only distantly related tomembers of the first major PspC family. FIG. 14 provides a dendrogramshowing the distance of this divergent PspC from the other PspCs. FIG.15 provides the amino acid and DNA sequences of the divergent PspC.

Inventive compositions, such as immunogenic, immunological or vaccinecompositions, can comprise at least one PspC (or immunogenic fragmentthereof or polypeptide comprising at least one PspC epitope or epitopicregion or at least one vector or plasmid expressing such PspC orfragment thereof, or at least one PspC epitope or epitopic region),preferably at least two (2), for instance up to ten (10), from strainsfrom each clade, alone, or in further combination with at least one PspA(or immunogenic fragment thereof or polypeptide comprising at least onePspA or at least one epitope or epitopic region of PspA or at least onevector or plasmid expressing such PspA or fragment thereof, or at leastone PspA epitope or epitopic region, which vector or plasmid can be thesame as the aforementioned vector or plasmid) or preferably at least two(2), for instance up to ten (10), from strains from each PspA clade, fora broadly efficacious pneumococcal vaccine with a limited number ofstrains.

Accordingly, in an aspect, the invention provides an immunogenic,immunological or vaccine composition containing an epitope of interestfrom at least one PspC and/or PspA, and a pharmaceutically acceptablecarrier or diluent. An immunological composition elicits animmunological response—local or systemic. The response can, but need notbe, protective. An immunogenic composition likewise elicits a local orsystemic immunological response which can, but need not be, protective.A vaccine composition elicits a local or systemic protective response.Accordingly, the terms “immunological composition” and “immunogeniccomposition” include a “vaccine composition” (as the two former termscan be protective compositions).

The invention therefore also provides a method of inducing animmunological response in a host mammal comprising administering to thehost an immunogenic, immunological or vaccine composition. From thedisclosure herein and the documents cited herein, including theapplications cited under “Related Applications”, the skilled artisan canobtain an epitope of interest of PspC and/or PspA, without undueexperimentation.

Further, the invention demonstrates that more than one serologicallycomplementary PspC molecule can be in an antigenic, immunological orvaccine composition, so as to elicit better response, e.g., protection,for instance, against a variety of strains of pneumococci; and, theinvention provides a system of selecting PspCs for a multivalentcomposition which includes cross-protection evaluation so as to providea maximally efficacious composition.

The determination of the amount of antigen, e.g., PspC or truncatedportion thereof or a polypeptide comprising an epitope or epitopicregion of PspC, and optional adjuvant in the inventive compositions andthe preparation of those compositions can be in accordance with standardtechniques well known to those skilled in the pharmaceutical orveterinary arts.

In particular, the amount of antigen and adjuvant in the inventivecompositions and the dosages administered are determined by techniqueswell known to those skilled in the medical or veterinary arts takinginto consideration such factors as the particular antigen, the adjuvant(if present), the age, sex, weight, species and condition of theparticular patient, and the route of administration.

For instance, dosages of particular PspC antigens for suitable hosts inwhich an immunological response is desired, can be readily ascertainedby those skilled in the art from this disclosure, as is the amount ofany adjuvant typically administered therewith. Thus, the skilled artisancan readily determine the amount of antigen and optional adjuvant incompositions to be administered in methods of the invention. Typically,an adjuvant is commonly used as 0.001 to 50 wt % solution in phosphatebuffered saline, and the antigen is present on the order of microgramsto milligrams, such as about 0.0001 to about 5 wt %, preferably about0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt %(see, e.g., Examples below or in applications cited herein).

Typically, however, the antigen is present in an amount on the order ofmicrograms to milligrams, or, about 0.001 to about 20 wt %, preferablyabout 0.01 to about 10 wt %, and most preferably about 0.05 to about 5wt %.

Of course, for any composition to be administered to an animal or human,including the components thereof, and for any particular method ofadministration, it is preferred to determine therefor: toxicity, such asby determining the lethal dose (LD) and LD₅₀ in a suitable animal model,e.g., rodent such as mouse; and, the dosage of the composition(s),concentration of components therein and timing of administering thecomposition(s), which elicit a suitable immunological response, such asby titrations of sera and analysis thereof for antibodies or antigens,e.g., by ELISA and/or RFFIT analysis. Such determinations do not requireundue experimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the time for sequentialadministrations can be ascertained without undue experimentation.

Examples of compositions of the invention include liquid preparationsfor orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric,mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratorymucosa) etc., administration, such as suspensions, syrups or elixirs;and, preparations for parenteral, subcutaneous, intradermal,intramuscular or intravenous administration (e.g., injectableadministration), such as sterile suspensions or emulsions. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient, such as sterile water, physiological saline, glucose or thelike. The compositions can also be lyophilized. The compositions cancontain auxiliary substances, such as wetting or emulsifying agents, pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Compositions of the invention, are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions which may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventioncan be in the “solid” form of pills, tablets, capsules, caplets and thelike, including “solid” preparations that are time-released or that havea liquid filling, e.g., gelatin covered liquid, whereby the gelatin isdissolved in the stomach for delivery to the gut. If nasal orrespiratory (mucosal) administration is desired, compositions may be ina form and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or,a dose having a particular particle size.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally. The viscous compositions may be in theform of gels, lotions, ointments, creams and the like and will typicallycontain a sufficient amount of a thickening agent so that the viscosityis from about 2500 to 6500 cps, although more viscous compositions, evenup to 10,000 cps, may be employed. Viscous compositions have a viscositypreferably of 2500 to 5000 cps, since above that range they become moredifficult to administer. However, above that range, the compositions canapproach solid or gelatin forms which are then easily administered as aswallowed pill for oral ingestion.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally, to animals, children, particularly small children,and others who may have difficulty swallowing a pill, tablet, capsule orthe like, or in multi-dose situations. Viscous compositions, on theother hand, can be formulated within the appropriate viscosity range toprovide longer contact periods with mucosa, such as the lining of thestomach or nasal mucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the antigen,lipoprotein and optional adjuvant. Minor amounts of other ingredients,such as pH adjusters (e.g., a base such as NaOH), emulsifiers ordispersing agents, buffering agents, preservatives, wetting agents,jelling agents (e.g., methylcellulose), colors and/or flavors may alsobe present. The compositions can be isotonic, i.e., it can have the sameosmotic pressure as blood and lacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount thatwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives, including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride, may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions must be selected to be chemically inert with respect to thePspC antigen and optional adjuvant. This will present no problem tothose skilled in chemical and pharmaceutical principles, or problems canbe readily avoided by reference to standard texts or by simpleexperiments (not involving undue experimentation), from this disclosureand the documents cited herein.

The immunologically effective compositions of this invention areprepared by mixing the ingredients following generally acceptedprocedures. For example the selected components may be simply mixed in ablender, or other standard device to produce a concentrated mixturewhich may then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity. Generally the pHmay be from about 3 to 7.5. Compositions can be administered in dosagesand by techniques well known to those skilled in the medical andveterinary arts taking into consideration such factors as the age, sex,weight, and condition of the particular patient or animal, and thecomposition form used for administration (e.g., solid vs. liquid).Dosages for humans or other mammals can be determined without undueexperimentation by the skilled artisan, from this disclosure, thedocuments cited herein, and the Examples below (e.g., from the Examplesinvolving mice and from the applications cited herein, e.g., under“Related Applications”, especially since PspC can be administered in amanner and dose analogous to PspA).

Suitable regimes for initial administration and booster doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, including applications cited herein, and theExamples below. The compositions can be administered alone, or can beco-administered or sequentially administered with other compositions ofthe invention or with other prophylactic or therapeutic compositions.Given that PspC is PspA-like, the skilled artisan can readily adjustconcentrations of PspA in compositions comprising PspA or a portionthereof to take into account the presence of PspC or a portion thereofin accordance with the herein teachings of compositions comprising atleast one PspC or portion thereof and optionally at least one PspA or aportion thereof.

The PspC antigen (PspC or a portion thereof), as well as a PspA antigen(PspA or a portion thereof) can be expressed recombinantly, e.g., in E.coli or in another vector or plasmid for either in vivo expression or invitro expression. The methods for making and/or administering a vectoror recombinant or plasmid for expression of PspC or a portion thereofeither in vivo or in vitro can be any desired method, e.g., a methodwhich is by or analogous to the methods disclosed in: U.S. Pat. Nos.4,603,112; 4,769,330; 5,174,993; 5,505,941; 5,338,683; 5,494,807;4,722,848; WO 94/16716; WO 96/39491; Paoletti, “Applications of poxvirus vectors to vaccination: An update,” Proc. Natl. Acad. Sci. USA93:11349–11353, (1996); Moss, “Genetically engineered poxviruses forrecombinant gene expression, vaccination, and safety,” Proc. Natl. Acad.Sci. USA 93:11341–11348, (1996); U.S. Pat. No. 4,745,051 (recombinantbaculovirus); Richardson, C. D. (Editor), Methods in Molecular Biology39, “Baculovirus Expression Protocols” (1995 Humana Press Inc.); Smithet al., “Production of Huma Beta Interferon in Insect with a BaculovirusExpression Vector,” Molecular and Cellular Biology, Vol. 3, No. 12, p.2156–2165 (1983); Pennock et al., “Strong and Regulated Expression ofEscherichia coli B-Galactosidase in Infect Cells with a Baculovirusvector,” Molecular and Cellular Biology Vol. 4, No. 3, p. 399–406(1984); EPA 0 370 573; U.S. patent application Ser. No. 920,197, filedOct. 16, 1986; EP Patent publication No. 265785; U.S. Pat. No. 4,769,331(recombinant herpesvirus); Roizman, “The function of herpes simplexvirus genes: A primer for genetic engineering of novel vectors,” Proc.Natl. Acad. Sci. USA 93:11307–11312, (1996); Andreansky et al., “Theapplication of genetically engineered herpes simplex viruses to thetreatment of experimental brain tumors,” Proc. Natl. Acad. Sci. USA93:11313–11318, (1996); Robertson et al., “Epstein Barr virus vectorsfor gene delivery to b lymphocytes,” Proc. Natl. Acad. Sci. USA93:11334–11340, (1996); Frolov et al., “Alphavirus based expressionvectors: Strategies and applications,” Proc. Natl. Acad. Sci. USA93:11371–11377, (1996); Kitson et al., J. Virol. 65, 3068–3075, 1991;U.S. Pat. Nos. 5,591,439 and 5,552,143 (recombinant adenovirus);Grunhaus et al., “Adenovirus as cloning vectors,” Seminars in Virology(Vol. 3) p. 237–52, (1992); Ballay et al. EMBO Journal, vol. 4, p.3861–65 (1993); Graham, Tibtech 8, 85–87, 1990; Prevec et al., J. Gen.Virol. 70, 429–434; PCT WO91/11525, Felgner et al. J. Biol. Chem. 269,2550–2561 (1994); Science, 259:1745–49, (1993); McClements et al.,“Immunization with DNA vaccines encoding glycoprotein D or glycoproteinB, alone or in combination, induces protective immunity in animal modelsof herpes simplex virus-2 disease,” PNAS USA 93:11414–11420, (1996), andU.S. Pat. Nos. 5,591,639, 5,589,466, and 5,580,859 relating to DNAexpression vectors, inter alia. See also, WO 98/335,10; Ju et al.,Diabetologia, 41:736–739, 1998 (lentiviral expression system); Sanfordet al., U.S. Pat. No. 4,945,050 (method for transporting substances intoliving cells and tissues and apparatus therefor); Fischbach et al.,(Intracel), WO 90/01543 (method for the genetic expression ofheterologous proteins by cells transfected); Robinson et al., seminarsin IMMUNOLOGY, vol. 9, pp. 271–283 (1997) (DNA vaccines); Szoka et al.,U.S. Pat. No. 4,394,448 (method of inserting DNA into living cells); andMcCormick et al., U.S. Pat. No. 5,677,178 (use of cytopathic viruses fortherapy and prophylaxis of neoplasia).

The expression product generated by vectors or recombinants in thisinvention optionally can also be isolated and/or purified from infectedor transfected cells; for instance, to prepare compositions foradministration to patients. However, in certain instances, it may beadvantageous to not isolate and/or purify an expression product from acell; for instance, when the cell or portions thereof enhance the effectof the polypeptide.

An inventive vector or recombinant expressing PspC or a portion thereofand/or PspA or a portion thereof can be administered in any suitableamount to achieve expression at a suitable dosage level, e.g., a dosagelevel analogous to the aforementioned dosage levels (wherein the antigenor epitope of interest is directly present). The inventive vector orrecombinant can be administered to a patient or infected or transfectedinto cells in an amount of about at least 10³ pfu; more preferably about10⁴ pfu to about 10¹⁰ pfu, e.g., about 10⁵ pfu to about 10⁹ pfu, forinstance about 10⁶ pfu to about 10⁸ pfu. In plasmid compositions, thedosage should be a sufficient amount of plasmid to elicit a responseanalogous to compositions wherein PspC or a portion thereof and/or PspAor a portion thereof are directly present; or to have expressionanalogous to dosages in such compositions; or to have expressionanalogous to expression obtained in vivo by recombinant compositions.For instance, suitable quantities of plasmid DNA in plasmid compositionscan be 1 μg to 100 mg, preferably 0.1 to 10 mg, e.g., 500 micrograms,but lower levels such as 0.1 to 2 mg or preferably 1–10 μg may beemployed. Documents cited herein regarding DNA plasmid vectors may beconsulted for the skilled artisan to ascertain other suitable dosagesfor DNA plasmid vector compositions of the invention, without undueexperimentation.

Returning to our discussion of the examples and results presentedherein, a rabbit polyclonal serum to PspC was made by immunization witha recombinant truncated clade b allele. The serum reacted with both PspCand PspA from fifteen (15) pneumococcal isolates indicating that PspCand PspA share extensive cross-reactive epitopes. The cross-reactiveantibodies appeared to cause cross-protection in a mouse model system.Mice immunized with recombinant clade B PspC were protected againstchallenge with a strain that expressed PspA but not PspC. In thisexperiment, the PspA-PspC cross-reactive antibodies were directed to theproline-rich domain present in both molecules.

More in particular, S. pneumoniae possess a family of proteins that bindphosphocholine (Brooks-Walter et al. 1997; Garcia et al. 1986; McDanielet al. 1992) present in the teichoic acid and the lipoteichoic acid ofthe cell membrane and the cell wall (Tomasz 1967). The choline-bindingproteins of pneumococci and other Gram-positive organisms all containstructurally similar choline-binding domains, which are composed ofmultiple tandem amino acid repeats (Breise et al. 1985). Autolysin, PspA(pneumococcal surface protein A), and PcpA (pneumococcal choline-bindingprotein A) of S. pneumoniae, toxins A and b of Clostridium difficile,glucosyltransferases from Streptococcus downei and Streptococcus mutans,CspA of Clostridium acetobiltylicum, and PspA of Clostridium perfringensall contain similar regions (Sanchez-Beato et al. 1995; Banas et al.1990; Barroso et al. 1990; Dove et al. 1990; Garcia et al. 1986;Sanchez-Beato et al. 1998).

In PspA from S. pneumoniae, these choline-binding repeats areresponsible for the attachment of PspA to the surface of thepneumococcus (Yother et al. 1994). PspA molecules interfere withcomplement activation (Briles et al. 1997), slow clearance ofpneumococci from the blood of infected mice (McDaniel et al. 1987), andelicit protection against pneumococcal sepsis and nasal carriage(McDaniel et al. 1991; Wu et al. 1997). A single non-pspA locus has beenidentified which has greater similarity to the choline-binding andproline rich regions of pspA than any of the other choline-binding genes(McDaniel et al. 1992). Applicants have designated the molecule PspCbecause of its strong molecular and serologic similarities to PspA(Brooks-Walter et al. 1997; see also applications cited under RelatedApplications, supra, note that in those applications initially PspC wascalled “PspA-like”, and pspC was considered pspA-like).

Other PspA-like proteins and pspA-like loci, which could be the same asPspC and pspC, have also been characterized and sequenced (SpsA, whichreportedly binds secretory IgA, Hammerschmidt et al. 1997;choline-binding protein (for binding a moiety on eukaryotic surfaces),CbpA, Rosenow et. al. 1997; see, e.g., FIGS. 8, 9, 10). Immunizationwith a crude extract of pooled non-PspA choline-binding proteinscontaining CbpA elicited protection to a lethal challenge of pneumococciintroduced intraperitoneally into mice (Rosenow et al. 1997).

In the present studies, Applicants have demonstrated that immunizationwith purified PspC is able to elicit protection against sepsis, and thisprotection is apparently mediated by antibodies cross-reactive withPspA. Applicants have also examined the genetic diversity present withinthis genetic locus, herein called pspC, by the examination of 12sequenced alleles. These include the previously sequenced alleles ofcbpA and spsA, an allele from the genomic sequencing project, and sevennewly sequenced pspC genes presented here for the first time.

The sequences of cbpA and spsA both included sequences of D39 or itsderivatives. Rosenow et al. sequenced cbpA from LM91 a pspA-mutant ofD39 (Rosenow et al. 1997); and Hammerschmidt et al. sequenced spsA froman encapsulated derivative of R36A (ATCC 1733) (Hammerschmidt et al.1997; see also FIGS. 8, 9, 10). From a comparison of these twosequences, it was apparent that spsA sequence contained a 480 bpdeletion within the gene. Because of this discrepancy, Applicants alsoreported a sequence of pspC from a cloned HindIII-EcoRI chromosomalfragment of D39 that was determined prior to the cbpA and spsA sequence(Brooks-Walter et al. 1997; see also applications cited under RelatedApplications, supra). This sequence matched exactly that of cbpA. Othersequences that were used for sequence alignment comparisons included twospsA sequences from capsular serotype 1 and 47 strains (Hammerschmidt etal. 1997), and the pspC/cbpA/spsA sequence from the capsular serotype 4strain sequenced in the TIGR genome project.

The invention shall be further described by way of the followingExamples and Results, provided for illustration and not to be considereda limitation of the invention.

EXAMPLES AND RESULTS

Materials and Methods

Bacterial Strains, Plasmids, and recombinant DNA Techniques

Chromosomal DNA from S. pneumoniae EF6796, a serotype 6A clinicalisolate (Salser et al. 1993), and D39, a serotype 2 isolate, wasisolated using a cesium chloride gradient procedure. The HindIII-EcoRIfragment of EF6796 and D39 was cloned in a modified pZero vector(Invitrogen, San Diego, Calif.) in which the Zeocin-resistance cassettewas replaced by a kanamycin cassette, kindly provided by Randall Harris.Recombinant plasmids were electroporated into E. coli TOP10F′ cells [F′{lacI^(q)Tet^(R)} mcrA_(mrr-hsdRMS-mcrBC) f80lacZ_M15_lacX74 deoR recAlaraD139_(ara-leu)7697 galU galK rpsL endA1 nupG] (Invitrogen). DNA waspurified from agarose using Gene Clean (Bio101, Inc., Vista, Calif.).

Chromosomal DNA used for PCR was isolated using a chloroform-isoamylalcohol procedure. Oligonucleotide primers, ABW13 (5′CGACGAATAGCTGAAGAGG 3′) (SEQ ID NO: 76) and SKH2(5′CATACCGTTTTCTTGTTTCCAGCC 3′) (SEQ ID NO: 77), were used to amplifythe DNA encoding the alpha-helical region and the proline-rich region ofpspC in 100 additional S. pneumoniae strains. These primers correspondto nucleotides 215–235 and nucleotides 1810–1834, respectively, of thepspC/EF6796 gene. PCR products from L81905 (serotype 4), BG9163(serotype 6B), DBL6A (serotype 6A), BG8090 (serotype 19) and E134(serotype 23) were cloned into pGem (Promega) or Topo TA vector(Invitrogen) which utilize the A overhangs generated by Taq polymerase.

Sequencing and DNA Analysis

Sequencing of pspC was completed using automated DNA sequencing (ABI377, Applied Biosystems, Inc., Foster City, Calif.). Sequence analyseswere performed using the University of Wisconsin Genetics Computer Group(GCG) programs (Devereux et al. 1984), MacVector 6.5 (Oxford Molecular),Sequencer 3.0 (GeneCodes, Inc.), and DNA Strider programs (Salser et al.1993). Sequence similarities of pspC were determined using the NCBIBLAST. Coil structure predicted by the pspC sequence was analyzed usingMatcher (Fischetti et al. 1993). The accession number by Genbank/EMBLfor the nucleotide sequences of PspC are as follows: EF6796-U72655,DBL6A-AF068645, D39-AF068646, E134-AF068647, BG8090-AF068648,L81905-AF068649, BG9163-AF068650, DBL6A-AF068645, D39-AF068646,E134-AF068647, BG8090-AF068648, L81905-AF068649, and BG9163-AF068650;and each of these sequences and GenBank results from the accessionnumbers are hereby expressly incorporated herein by reference (See alsoFIGS. 11 and 15–21). Preliminary sequence data was obtained from TheInstitute for Genomic Research website.

Deposit

E. coli containing a cloned PspC gene from Streptococcus pneumoniaestrain EF6796 was deposited on Jul. 24, 2001 with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110–2209, USA, under accession number ATCC No. PTA-3526.

Example/Result 1 Sequence Analysis of pspC Gene-Aspects Relating toDomain Structure and Function

The protein sequences of pspC, spsA, and cbpA were aligned usingMacVector 6.5 (FIGS. 1, 2, and 13). The predicted amino acid sequencesencode proteins ranging in size from 59 to 105 kDa protein. The signalsequences of 37 amino acids are highly conserved (84–100% Identity). Themajor part of each protein is composed of a large alpha-helical domain(FIGS. 1, 2, and 13). The N-terminal 100 to 150 amino acids of thisalpha-helical domain are hypervariable in both size and sequence and areunique for each strain sequence of unrelated parentage (FIG. 2, D39,SpsA2, CbpA, and Cb3P are all from a related lineage; see also FIG. 13).In the hypervariable regions of capsular serotype 1 and 4 strains, thereis a unique 23 amino acid serine-rich sequence (amino acid positions 112to 135).

Downstream of the hypervariable region and central to the alpha-helicaldomain is the first of two direct repeats. The amino acid repeats (FIGS.2, 13) vary in size in individual PspCs from 101 to 205 amino acids andare approximately 79–89% identical at the amino acid level.Smaller-sized amino acid repeats in some strains differ from the largerrepeats of other strains only by lack of sequence at the NH₂-terminalend, which accounts for their smaller size. The first repeat in eachstrain is more like the corresponding first repeat of other strains thanit is like the second repeat of the same strain. This pattern suggeststhat duplication forming this repeat happened in an ancestral gene,prior to the diversification of pspC into the numerous divergent allelesseen today. These repeats are highly charged with approximately 45% oftheir sequence being either lysine or glutamic acid residues. Thesealpha-helical repeats were present in all alleles that were examinedexcept for the spsA//serotype 1 and spsA//serotype 2 (Hammerschmidt etal. 1997) (FIGS. 2, 13).

Between the amino acid repeats of the alpha-helical domain is a highlyconserved 40 amino acid sequence break in the coiled-coil motif whichwas identified using the Matcher program (Fischetti et al. 1993) (FIGS.2, 13 and 3). Matcher examines the characteristic seven residueperiodicity of coiled-coil proteins arising largely from thepredominance of hydrophobic residues in the first and fourth positions(a and d) and non-hydrophobic residues in the remaining positions(Fischetti et al. 1993). The coiled-coil region of the alpha-helix ofPspC/EF6796 has three breaks in the heptad repeat motif (FIG. 3). Theseinterruptions of the heptad motif in the 7-residue periodicity wererespectively 6, 44 and 5 amino acids in length. Similar breaks atcorresponding sequence positions were found in all PspC alleles.

In some molecules of PspC, the proline-rich region followed the secondamino acid repeat (FIGS. 1, 2, and 13). However, in the three largerPspC molecules, a region very similar to a corresponding region of thepspA genetic locus is present. Based on whether this pspA-like regionwas present or absent and on a distance-based cluster analysis, PspCmolecules were classified into two clades (FIGS. 4, 12). Clade Amolecules contained the pspA-like element and were larger in size. PspCclade b molecules were smaller and lacked this pspA-like region. ThispspA-like region (alpha-helical-2) was present in PspC/BG9163, EF6796and BG7322 (FIGS. 1, 2, and 13 and Table 1) as well as in many pspAgenes.

Although there is some variation within the proline-rich region of thesequenced PspCs (FIGS. 1, 2, 13), the region is not distinguishable fromthe proline-rich region of PspA molecules. Within PspA molecules, twotypes of proline-rich regions have been identified. One type, whichcorresponds to about 60% of PspAs, contains a central region of 27non-proline amino acids, which is highly conserved. The other type ofproline-rich region in PspA lacks this conserved non-proline region. Inthe case of PspC, clade A strains lacked the 27 amino acidnon-proline-rich block, whereas the four clade b PspC molecules had thisconserved block. When present, the sequence of the 27 amino acidnon-proline-rich region is highly conserved between PspC and PspAmolecules. No correlation was observed between the expression of thisconserved region within PspA and PspC molecules produced by the samestrain. The proline-region of SpsA/serotype 1 was different from that ofall other PspC molecules. This proline-rich region of this SpsA moleculehas a truncated proline-rich region, which contains the 27 amino acidnon-proline break but lacks the NH₂ end of the proline-rich region.

The choline-binding repeat domains of PspC, CbpA and SpsA proteins eachcontain between 4 and 11 repeats of about 20 amino acids (FIG. 5). Therepeats found in the center of the choline-binding domain were closestto the consensus sequence, while repeats on the NH₂-terminal andCOOH-terminal ends of the block were more distant from the consensussequence. The arrangement of repeats over the entire choline-bindingregion in PspC was examined relative to the arrangement of similarrepeats in the choline-binding region of five PspC and three PspA genesfor which the entire choline-binding domain was sequenced. The followingfindings all suggested a very close relationship between PspA and PspCin the choline-binding region of the molecule: 1) the NH₂-terminaldivergent repeat is identical between the paralogous proteins (PspA andPspC); 2) similarly, the COOH-terminal divergent repeats are verysimilar between PspC and PspA (see repeats 10 and 11 of PspC consensusand repeats 9 and 10 of PspA consensus—FIG. 5), yet these repeats arehighly diverged from the rest of the repeat block; 3) the conservedcentral repeats of the choline-binding domain in each case have a singleamino acid at position 6 which is frequently asparagine in PspC, butusually tyrosine in PspA proteins (other than position 6, the consensusrepeat for both genes is identical); 4) divergence of individual aminoacids within the 20 amino acid repeat from the repeat consensus sequencewas identical between PspA and PspC (position number 4, 6, 9, 12, 13,15, 16, and 18); and 5) the repeat blocks are followed by a 17 aminoacid partially hydrophobic “tail” that is nearly identical for PspC orPspA except for an additional asparagine present at the end of the PspCproteins that is missing from PspA proteins. Overall, thecholine-binding domains of PspA and PspC are so similar that it wouldnot be possible to determine with certainty whether any particularcholine-binding domain from these two proteins belongs to PspA or PspCwithout knowledge of its flanking DNA.

Example/Result 2 Phylogenetic Analysis

The pspA and pspC genes are paralogs of each other because they are bothpresent in the genome of most pneumococci, and because they share highidentity in the sequence encoding their COOH-terminal halves (Table 1).An alignment of the 12 PspC/CbpA/SpsA sequences was constructed usingthe Clustal W algorithm (FIGS. 2, 13). An unrooted phylogram wasproduced with PAUP 4.0B with the neighbor-joining method from the meanamino acid distances as calculated over this alignment (FIGS. 4, 12).The figure as shown incorporates both distance measurements along thebranch lengths and bootstrap analysis of 1000 repetitions. Branch lengthbetween molecules is proportional to the similarity of the sequences.The tree represents the evolutionary hypothesis that PspC moleculesarose in two main clusters representing clades A and B. One clade, A,consisted of the larger PspC molecules, and contained strong identity inalpha-helical region-2 with some pspA alleles. The second clade, B, didnot contain this region of identity with pspA alpha-helical regionpspAs.

Example/Result 3 Analysis of pspC Using PCR

PCR was used to amplify pspC from different strains of S. pneumoniae topermit studies of the variability of PspC. Two oligonucleotides whichrecognized the common sequence regions of pspC, but which did notamplify the pspA genes, were designed in an effort to permit specificamplification of pspC alleles from all pneumococcal strains.Oligonucleotide ABW13 is specific to DNA upstream of the promotersequence of the pspC gene locus. Oligonucleotide SKH2 is specific to theDNA encoding the C-terminal end of the proline-rich region of both thepspA and pspC gene loci. These oligonucleotides were used to amplifyfragments of pspC from 100 S. pneumoniae strains. Seventy-eight of the100 strains produced PCR-generated fragments, which varied from 1.5 kbto 2.2 kb in size. The remaining 22 strains failed to produce a PCRproduct. Based on the strains of known sequence it was observed that thesize of the amplified products correlated with whether they were clade Aor clade B. Because of the absence of this pspA-conserved region, theclade b pspC sequences were smaller than the clade A pspC. The amplifiedproduct using oligonucleotide ABW13 and SKH2 of clade A molecules was2.0 kb or greater. The amplified fragment of clade b molecules wasapproximately 1.6 kb. Approximately 4% of the 75 strains from which apspC gene was amplified were found to be clade A by this criterion and96% were clade B.

Example/Result 4 Cloning and Expression of a Recombinant Truncated PspCMolecules

Oligonucleotides were used to amplify a 1.2 kb fragment of L81905, whichencodes amino acids 263–482 of the alpha-helix and proline-rich regionof PspC. The amplified PCR fragment was cloned into pQE40 (Qiagen,Chatsworth, Calif.) which allows expression of a fusion product with apolyhistidine tag at the amino-terminal end, followed by dihydrofolatereductase (DHFR), and then by the fragment of PspC/L81905 (263–482).Expression of the fusion protein in E. coli strain BL21(DE3) was inducedduring growth at room temperature by the addition of 1 mMisopropyl-b-d-thiogalactopyranoside (IPTG). The overexpressed fusionprotein was purified by affinity chromatography under non-denaturingconditions over a nickel resin according to the manufacturer'sprotocols. Purified fusion protein was then analyzed by SDS-PAGE andquantitated using a BioRad Protein Assay (Hercules, Calif.). Twofragments of PspC/D39 (AA 1–445 and AA 255–445), and three fragments ofPspA/Rx1 (AA 1–301, AA 1–314 and AA 1–370) were expressed as fusionproteins with 6×His tag in the pET20b expression system (Novagen,Madison, Wis.). In this case, the overexpressed fusion proteins containa PelB leader peptide, followed by the PspC or PspA fragments and theHis tag at the carboxy-terminus. Expression was induced for pET20b-basedconstructs with 0.4 mM IPTG in the expression strain BL21(DE3), andpurified according the manufacturer's protocol.

Example/Result 5 Production of a Polyclonal Antiserum, SDS-PAGE, andImmunoblots

The truncated product (AA 263 to 482) of PspC/L81905 was purified bymetal affinity chromatography and used to immunize a rabbit.Approximately 4 μg of purified PspC from L81905 was injected two timessubcutaneously into a rabbit twice on consecutive weeks and blood wascollected 10 days after the last injection. The primary immunization waswith Freund's complete adjuvant and the booster immunization was givenin saline. Polyclonal rabbit antiserum was diluted 1:50 and used toanalyze pneumococcal lysates on a 7.5% SDS-PAGE gel (BioRad, Hercules,Calif.). Pneumococcal lysates and immunoblots were performed asdescribed by Yother et al. 1994.

Example/Result 6 Cross-Reactivity of Antisera Made to PspC/L81905 withPspA and Other PspC Molecules

A truncated product (AA 263–482) of the PspC/L81905 clade b pspC proteinwas expressed in E. coli using the Qiagen Expression system. It shouldbe noted that PspC/L81905 is clade b and lacks the pspA-like region inits alpha-helix. The truncated (AA 263–482) clade b PspC protein waspurified by metal affinity chromatography and used to immunize a rabbitto generate a polyclonal antiserum to PspC. Pneumococcal lysates wereseparated on SDS-polyacrylamide gels and blotted to nitrocellulose. Theblots were developed either with Xi126, a monoclonal antibody to PspA,or with the anti-PspC rabbit polyclonal antiserum. The reactivity of thePspC antiserum with selected pneumococcal lysates run in a Westernimmunoblot is shown in FIG. 6.

The reactivity pattern of the antiserum to PspC was deciphered in partusing lysates from S. pneumoniae strains JY1119 and JY53. These strainsare derivatives of the pneumococcal strains WU2 and D39, respectively,in which the pspA genes have been insertionally inactivated (Yother etal 1992). From the Western blot, it is apparent that the polyclonalserum reacts with a 90 kDa band in JY53 even though the pspA gene hasbeen inactivated in this strain. This band is assumed to represent PspC.Both JY1119 and its parent, WU2, lack the pspC gene altogether (McDanielet al. 1992). An 85 kDa molecule from WU2 reacts with the anti-PspCantiserum and with the anti-PspA MAb. This band is not present inJY1119, which contains an insertionally inactivated PspA.

The rabbit antiserum was reactive with proteins in the lysates from allpneumococcal strains tested. The relative molecular weights of theproteins detected also made it apparent that the antiserum was reactingwith both PspA and PspC molecules. To distinguish cross-reactivity withthe PspA molecule from direct reactivity with the PspC molecule inuntested strain lysates a second identical Western blot was developedwith a monoclonal antibody specific to PspA molecules (FIG. 6, part B).PspC bands could be identified through the comparison of bandingpatterns in parts A and b of FIG. 6. The bands reactive with theanti-PspC rabbit antiserum but not with the anti-PspA MAb wereidentified as PspC. Bands stained by the rabbit antiserum thatco-migrate with those also stained by the MAb were PspA molecules thatcross-reacted with the antiserum to PspC. Besides failing to react withthe MAb, it was also noted that PspC bands were of higher molecularweight than the PspA bands. By these criteria the anti-PspC serumcross-reacted with PspA in all strains tested except A66. For A66, asingle band was detected. Further testing determined this band to bePspA-derived, rather than PspC-derived. In this case, A66 lacked a pspCgene and the PspA of A66 was not reactive with the MAb used, Xi 126,even though anti-PspA immune sera does detect PspA in this strain. Fromthe above patterns of reactivity, it was concluded that the PspCpolyclonal antiserum is cross-reacting specifically with the PspAmolecule.

Example/Result 7 Immunization and Challenge Studies

CBA/N mice were immunized with purified recombinant PspC proteinsoriginating from strain L81905 (AA 263–482), the full alpha-helicalregion of PspC in strain D39 (AA 1–445), or a truncated portion of thePspC protein in strain D39 (AA 255–445). Each mouse received only one ofthe above recombinant proteins and groups of 5–6 mice were immunized ineach experiment. The mice were immunized subcutaneously withapproximately 1 μg of purified protein emulsified in 0.2 ml of completeFreund's adjuvant. Three weeks later they were boosted with 1 μg ofpurified protein in saline. Three weeks after the boost, the mice werechallenged with approximately 700 colony-forming units (CFU) ofpneumococcal strain WU2. Control mice were immunized with buffer andcomplete Freund's adjuvant without PspC.

Analysis of Immune Sera: Mice were bled retroorbitally 24 hours beforechallenge. The blood was collected into 0.5 ml 1% BSA/phosphate bufferedsaline. Samples were centrifuged for 1 min (2000 rpm) and thesupernatant was collected and stored at −20° C. until used in directELISAs (enzyme-linked immunosorbent assays). Microtiter 96 well plates(Nunc, Weisbaden, Germany) were coated overnight at 4° C. with 0.5 μg ofexpressed protein which included PspC (AA 1–445) and PspA (UAB55-AA1–301, UAB15-AA 1–314 and UAB103-AA 1–370). Plates were blocked with 1%bovine serum albumin/phosphate buffered saline (PBS) followed byincubation with immune sera for 3 hours at 37° C. Plates were washedwith PBS/DAKO with 0.15% Tween and incubated with goat anti-mouseimmunoglobulin biotin-conjugated antiserum and streptavidin alkalinephosphatase (Southern Biotechnology Assoc., Birmingham Ala.). They weredeveloped with p-nitrophenyl phosphate (Sigma, St. Louis, Mo.). The logreciprocal titer giving 33% maximum binding to the mouse immune sera wasdetermined to evaluate the reactivity.

Ability of PspC to elicit protective immunity in mice: Mice wereimmunized with one of three purified fragments of clade b PspC: L81905(AA 263–482), D39 (AA 1–445) and D39 (AA 255–445). None of theseimmunogens contained the pspA-like alpha-helical region 2 noted earlier,but all of the immunogens contained the proline-rich region. Miceimmunized with PspC and control mice then immunized with adjuvant onlywere challenged with WU2 or BG7322. WU2 is a capsular serotype 3 strainthat produces no detectable PspC and does not contain the structuralgene for pspC (FIG. 6). BG7322 is a capsular serotype 6b strain andcontains a clade A PspC molecule. Significant protection against deathwas seen with both challenge strains in mice immunized with the threedifferent PspC clade B molecules (Table 2). Protective immunity in micechallenged with WU2 must derive from the ability of the PspC immunogento elicit immunity (presumably mediated by antibodies) in the mice thatcross-reacts with the PspA molecule present on surface of strain WU2because this strain lacks PspC. The ability of PspC to elicit immunitythat is directed against PspA was expected from the data herein sincePspC had been shown to elicit antibodies cross-reactive with PspA (FIG.6). Protection of the mice challenged with BG7322 was statisticallysignificant even though only 62% of the mice were protected as opposedto 96% when challenged with WU2.

Example/Result 8 Antibody Elicited to Recombinant PspC

For this study sera was used from mice immunized with LXS240, whichencoded amino acids 255–445 of clade b PspC/D39. This sequence containsthe entire proline-rich region of PspC/D39. Direct binding ELISAs wereconducted to localize the epitope yielding the cross-reactivity withPspA. Microtiter 96 well plates were coated with fragments of PspC/D39and PspA/Rx1. Each of the cloned PspA/Rx1 molecules used in these assaysexpressed the PspA alpha-helical region and differed only in the numberof the amino acids it contained in the proline-rich region. UAB55contained 15 amino acids in the proline-rich region, UAB15 contained 26amino acids in the proline-rich region, and UAB103 contained the entireproline-rich region. The results from the ELISA are depicted in FIG. 7.Mouse antisera only reacted with the PspA/Rx1 molecules containing theentire proline-rich region. The antisera did not react with the PspAmolecules UAB55 and UAB15 that contained truncated proline regions.These results strongly suggest that the antibodies elicited by PspC thatcross-protect against PspA are probably directed at the proline-richregions of these molecules. Accordingly, the invention comprehends amethod for eliciting anti-PspA antibodies comprising administering PspCor an epitopic region thereof or a polypeptide comprising an epitope ofPspC.

Example/Result 9 Modular Evolution and Chimeric Structure of pspC

PspC is a chimeric protein which has acquired domains from bothinterspecies and intraspecies genetic exchanges. The protein contains asignal sequence that has 75% nucleotide identity to the bac gene fromgroup b streptococci (accession numbers X59771 and X58470)(Hammerschmidt et al. 1997). The bac gene encodes the b antigen of Groupb streptococci, a cell surface receptor that binds the constant regionof human IgA. This similar sequence in the signal peptide regionsuggests that potential interspecies genetic exchange between group bstreptococci and S. pneumoniae may have formed a chimeric locusincluding the bac regulatory region and a partial pspA or a pspA-likelocus to create an ancestral gene for pspC. The origin of the centralregion specific to the current pspC genes is unknown. The direct aminoacid repeats of the alpha-helix suggest that this region of PspC hasevolved by a domain duplication event. This internal duplication of aportion of the alpha-helix led to gene elongation. The region of thealpha-helix is presumably the functional region of the molecule andreportedly binds SIgA (Hammerschmidt et al. 1997). Further intraspeciesvariation events are hinted at in the finding that 4% of PspC proteinsare of lade A. This clade appears to have derived from a recombinationevent with PspA (or visa versa) providing further evidence of chimericstructure of PspC and possibly PspA molecules.

Several functions have been attributed to the PspC molecule. In additionto binding secretory IgA and a moiety on the surface of epithelialcells, Hoistetter et al. have reported that PspC binds the complementcomponent C3 (Hostetter et al. 1997). Recent studies have shown thatPspA inhibits complement activation by inhibiting the formation of theC3 convertase. With the similar structural domains of PspA and PspC, itis conceivable that the virulence properties of the two proteins maycomplement each other in the host. WU2 is a strain of S. pneumoniae thatdoes not contain a structural gene for PspC. When mutants of PspA areproduced in WU2 that lacks PspC there is a 10,000-fold decrease invirulence (Briles et al. 1997). When PspA is mutated in D39, a strainthat contains both PspA and PspC, there is only a 10-fold decrease invirulence (Briles et al. 1997). From the data herein, PspA and PspC maycomplement each other in their abilities to block the clearance ofpneumococci by interfering with the complement pathway (see also thepreliminary data of Hostetter et al. 1997 and the data of Briles et al.1997).

Rosenow et al. demonstrated that CbpA is expressed more strongly bypneumococci in the nasopharynx than by pneumococci in the blood (Rosenowet al. 1997). Thus, it is feasible that the two molecules may serve thesame general function, possibly in different host tissues and indifferent stages of infection. Furthermore, either molecule may be morecritical to virulence in the absence of the other. This hypothesis isfurther strengthened by data from ongoing studies that show that mutantslacking in both PspC and PspA are significantly decreased in virulence.

In PspC immunization studies, Applicants challenged mice with a strainexpressing both PspC and PspA and a strain expressing PspA but not PspC.By including strains lacking the pspC gene Applicants could determine ifprotection elicited by PspC required the expression of PspC or mightact, at least in part, through cross-reactions with PspA. For the studypresented, mice were immunized with clade B PspC. This molecule lacksthe PspA-PspC homology region near the C-terminal end of thealpha-helical region of PspC. Thus, this immunogen was expected to beone that would give less cross-reaction with PspA than would a clade APspC. Even so, immunization with PspC/D39 resulted in protection whenmice were challenged either with strain BG7322, which expresses bothPspA and PspC, or with strain WU2, which expresses PspA but lacks PspC.

The protection-eliciting PspC immunogen contained the entireproline-rich region. The alpha-helical regions of PspA/WU2 and PspC/D39have essentially no homology. However, the proline-rich region of PspCis repetitive and homologous with PspA. It was possible that antibody tothis region was responsible for the cross-protection we observed. Thishypothesis was supported by the observation that antibody elicited toPspC reacted with PspA fragments that contained the proline-rich regionbut not with those that lacked the proline-rich region in direct ELISAs.Antibodies elicited by PspC also cross-reacted with PspA on Westernblots. The likelihood that the protective cross-reaction of PspC immunesera is mediated through PspA was further strengthened by the sequencedata released by TIGR. Extensive searches of the largely completedgenome failed to find other pneumococcal gene sequences with as high asimilarity with the PspC sequence domains as the proline-rich region ofPspA.

Electron microscopy surface labeling studies and epitope mapping studieshave localized PspA on the surface of pneumococci with the largelyexposed alpha-helical region (Gray, Pneumococcal infection, in BacterialInfection, P. E. Brachman, Ed. 1997, Plenum Pub. Corp. NY; McDaniel etal. 1994; McDaniel et al., Monoclonal antibodies against surfacecomponents of Streptococcus pneumoniae, in Monoclonal antibodies againstbacteria, A. J. L. Macario and E. C. de Macario, Eds. 1986, AcademicPress, Inc. Orlando). Studies by Yother and White have shown that PspAis attached by the C-terminal end to lipoteichoic acids (Yother et al.1994). No information has been available, however, about whether or notthe proline-rich domain is surface exposed. Results from theseexperiments indicating that antibodies to the proline-rich domain areprotective suggest that this domain of PspA is probably accessible onthe surface of the pneumococci. This study also provides the firstpublished evidence that antibodies reactive with the proline-rich regionof PspA can be protective against pneumococcal infection.

PspA, PspC/CbpA/SpsA, LytA and PcpA are proteins of S. pneumoniae thatcontain choline-binding domains. The choline-binding domains ofPspC/CbpA/SpsA contain between 4 and 11 repeats of about 20 amino acids.The consensus sequences of these repeats are from 90 to 95% identical.The middle region of the choline-binding domain of PspA and PspC isconserved. The first and last two repeats of PspA and PspC differsubstantially (by 40 to 65%) from the consensus sequence. Even so, PspAand PspC sequences in these areas generally have the same deviationsfrom the consensus sequence and in most cases are within 95% identical.The choline-binding domains of LytA and PcpA are quite different fromthat of PspA or PspC (42–62% identity) (Garcia et al. 1986;Sanchez-Beato et al. 1998). Whereas PspA and PspC have most likelyevolved by gene duplication, PcpA has probably arisen from horizontalgene transfer. The choline-binding regions of these proteins all supporta modular form of evolution of this group of proteins.

This disclosure provides a comprehensive study of the sequence of pspCand shows that PspCs can be divided into two clades based on thesequences in their alpha-helical and proline-rich domains. Thedisclosure also demonstrates that immunity to the proline-rich domain ofPspC can be protective through its recognition of the proline-richdomain of the PspA molecule. The fact that the N-terminal alpha-helicaldomain of PspC is different from the alpha-helical domain of PspAsuggests that PspC and PspA may serve somewhat distinct roles invirulence. However, the fact that the two molecules have a very similardomain structure and have similarity in much of their sequences raisesthe possibility that these two molecules may have similar functions.Although there are sequences of a few pspC alleles, this is the firstreport that the PspC family contains two clades and that the PspCmolecules contain homology to PspA within the cross-protective region ofthe alpha-helix. The identification of two clades of PspC is pertinentto PspC-containing vaccine, immunological or immunogenic compositions,as well as to methods for identifying PspA, pspA, PspC, pspC, and/or S.pneumoniae. Moreover, the observation that antibodies to theproline-rich regions of PspA and PspC can be cross-protectivefacilitates the design of more efficacious vaccines, as well as ofalternate vaccines, immunogenic or immunological compositions, e.g., byproviding epitopic regions of PspC, epitopes of PspC and nucleic acidmolecules encoding the same, and methods for identifying PspA, pspA,PspC, pspC, and/or S. pneumoniae.

TABLE 1 Conservation of PspC domains shown as percent amino acididentities. Clade A Clade B PspC vs. PspC vs. PspC vs. PspA vs. PspCPspA PspA PspA PspC Domain Orthologous Paralogous paralogous orthologousUpstream >97% No no >95% through signal alignment alignment Peptidepossible possible Whole gene 67.6–99%   14–29% 14–21% 22-79%Alpha-helical 1 66.9–99.6% 11.8–22.0% 14.8–23.1% not presentAlpha-helical 2 100% 13.1–88.7% not present 14–99% Proline-rich* High**high high high Choline-  87% 77% 79.1–99%   77–98% binding 17 AA tail100% 88.9% 88.9–94.4%  98–100% 3′ downstream 99% No no N.D. alignmentalignment possible possible Percentages calculated using a distancematrix from PAUP 3.0. *All PspA and PspC molecules have a repetitivesegment of protein in this region with the motif PEPK or PAPAP. Clade BPspC molecules have a conserved non-repetitive break in the proline-richregion. Distance ranges are uninformative because it is not possible toalign these sequences in a meaningful way.

TABLE 2 Cross-Protection of CBA/N Mice immunized with Recombinant PspCImmunogen Capsular Challenge non- Serotype strain and immunized²immunized² PspC of PspC Capsular # of mice # of mice fragment donorSerotype alive/dead³ alive/dead³ P value¹ L81905 4 WU2 (3) 13/0 1/12<.0001 (AA 263–248) D39 (AA 2 WU2 (3)  5/0 0/5  .008  1–445) D39 (AA 2WU2 (3)  4/1 0/5  .048 255–445) D39 (AA 2 BG7322 13/8 1/19 .0002255–445) (6B) ¹The statistical difference between immunized andnon-immunized was calculated using the Fisher exact test. ²Mice wereeither immunized with PspC with complete Freund's adjuvant or withadjuvant and buffer but no antigen. ³Mice were challenged 21 days postimmunization with 700 CFU of WU2 or 2000 CFU of BG7322 injected i.v. in0.2 Ringer's injection solution.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

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1. An isolated nucleic acid molecule encoding a pneumococcal surfaceprotein C (PspC) of S. pneumoniae comprising the nucleic acid sequenceselected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, SEQID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO:75.
 2. The nucleic acid molecule according to claim 1, wherein thenucleic acid molecule has the nucleotide sequence of SEQ ID NO:
 69. 3.The nucleic acid molecule according to claim 1, wherein the nucleic acidmolecule has the nucleotide sequence of SEQ ID NO:
 70. 4. The nucleicacid molecule according to claim 1, wherein the nucleic acid moleculehas the nucleotide sequence of SEQ ID NO:
 71. 5. The nucleic acidmolecule according to claim 1, wherein the nucleic acid molecule has thenucleotide sequence of SEQ ID NO:
 72. 6. The nucleic acid moleculeaccording to claim 1, wherein the nucleic acid molecule has thenucleotide sequence of SEQ ID NO:
 73. 7. The nucleic acid moleculeaccording to claim 1, wherein the nucleic acid molecule has thenucleotide sequence of SEQ ID NO:
 74. 8. The nucleic acid moleculeaccording to claim 1, wherein the nucleic acid molecule has thenucleotide sequence of SEQ ID NO:
 75. 9. A vector or plasmid comprisingthe isolated nucleic acid molecule according to claim 1.